All-optical observation on activity-dependent nanoscale dynamics of myelinated axons

Abstract. Significance In the mammalian brain, rapid conduction of neural information is supported by the myelin, the functional efficacy of which shows steep dependence on its nanoscale cytoarchitecture. Although previous in vitro studies have suggested that neural activity accompanies nanometer-scale cellular deformations, whether neural activity can dynamically remodel the myelinated axon has remained unexplored due to the technical challenge in observing its nanostructural dynamics in living tissues. Aim We aim to observe activity-dependent nanostructural dynamics of myelinated axons in a living brain tissue. Approach We introduced a novel all-optical approach combining a nanoscale dynamic readout based on spectral interferometry and optogenetic control of neural excitation in an acute brain slice preparation. Results In response to optogenetically evoked neuronal burst firing, the myelinated axons exhibited progressive and reversible spectral redshifts, corresponding to the transient swelling at a subnanometer scale. We further revealed that the activity-dependent nanostructural dynamics was localized to the paranode. Conclusions Our all-optical studies substantiate that myelinated axon exhibits activity-dependent nanoscale swelling, which potentially serves to dynamically tune the transmission speed of neural information.


Introduction
As neurons function by millisecond-scale ion flux across the cell membrane, neural activity has long been thought to accompany measurable morphological changes. [1][2][3] Since the late 1970s, several groups have reported nanometer-scale swelling in the giant axons of invertebrate species (crayfish and squid) by Michelson interferometry 4 and mechanoelectrical measurement. 5 Later studies in mammalian cultured neurons by dark-field microscopy and full-field interferometry revealed the subnanometer-scale morphological dynamics dependent on electrical potential across the neuronal cell membrane. [4][5][6][7][8] In contrast to the invertebrate models, the extent to which neural activity dynamically remodels the axons in living vertebrate brains has remained an enigma as these are orders-of-magnitude smaller and often ensheathed by the insulating layer of myelin. Myelin is a highly compacted subcellular structure of the oligodendrocyte, composed of multilayered lipid membranes and intervening aqueous mediums. This highly organized thin-film cytoarchitecture supports rapid and energy-efficient conduction of neural information in a small form factor, enabling formation of the highly integrated circuits of the mammalian brains. 9,10 Considering that the function of myelin has a steep dependence on its thin-film structure, the structural dynamics of myelinated axons, even at the subnanometer scale, can have a critical impact on neural circuit functions. 11 Investigating the nanostructural dynamics of myelinated axons in living mammalian brains is technically challenging. The current gold standard for nanoscale imaging of myelinated axons is electron microscopy, which is hardly applicable to living biological samples due to invasive sample preparation. [12][13][14] Super-resolution techniques are a promising alternative for living biological samples, but subnanometer-scale precision has yet to be attained in the mammalian axons due to high optical aberrations of the lipid-rich myelin layers. [15][16][17] Thus most studies have focused on long-term dynamics of relatively large morphological changes involving cell proliferation and differentiation. [11][12][13]18 Several years ago, we developed a spectral interferometric technique, named SpeRe, that offers the nanoscale readout of the thin-film cytoarchitecture of the myelinated axons in vivo. 19 Here we improved the SpeRe to have subnanometer spatial precision as well as subsecond temporal resolution and further combined it with optogenetic manipulation to unveil the neural activity-dependent nanostructural dynamics of myelinated axons in a living brain tissue. Our all-optical approach revealed for the first time that myelinated axons in a living mammalian brain exhibit subnanometer-scale swelling in response to neuronal burst firing and that the swelling dynamics is cumulative and reversible in the second scale.

Tapered Glass Fiber Sample
The tapered glass fibers were obtained by thermally drawing a glass rod (1.5-mm in diameter) using a micropipette puller (P-1000, Sutter Instrument). To mimic the typical axon diameters (1 to 5 μm), the optimal parameters for the micropipette puller were obtained by trial and error (ramp = 500 temperature = 550, pull = 40, velocity = 20, and pressure = 500). The tapered fiber was glued on a plain slide glass using an acrylic adhesive (401, Loctite) ensuring that the fiber was parallel to the slide glass (i.e., orthogonal to the optical axis of the objective lens). To firmly hold the fiber, a silicone-based sealant (Kwik-Cast, World Precision Instruments) was introduced around the fiber tip, exposing only the fiber tip in the air. The sample was imaged by a polarization microscope (SP8, Leica), and the diameter along the fiber was measured by applying the "distance map" module in ImageJ.

Artificial Cerebrospinal Fluid
Two types of artificial cerebrospinal fluid (aCSF) solutions were prepared; one is for surgery and the other is for recording. The surgical aCSF was composed of (in mM) 92 NMDG, 2.

Mouse Preparation
All mice were housed with littermates in groups of two to five in a reverse day/night cycle and given ad libitum access to food and water. All animal experiments were performed in compliance with institutional guidelines and approved by the subcommittee on research animal care at Sungkyunkwan University and Seoul National University. Male or female C57BL6J wild-type mice aged 4 weeks (Jackson Laboratory) were used for virus-mediated transduction of a genetically encoded calcium indicator (GCaMP6s) and/or an optogenetic protein (ChrimsonR-tdT). The mouse was anesthetized by inhaling 4% isoflurane (Hanapharm) in an induction chamber and was subsequently maintained with 1% to 1.5% isoflurane during surgery. The mouse skull was affixed on a custom-made stereotaxic frame and the body temperature was maintained at 37°C using a homeothermic blanket (TC-1000, CWE) and a thermistor probe (YSI-451, CWE). After removing the scalp, the hole was made on the center of the somatosensory area at a diameter of ∼1 mm. The 700-nL of a solution containing AAV9-hSyn-GCaMP6s and/or AAV5-hSyn-Chrimson-tdTomato (∼5 × 10 11 GC · ml −1 each in the recording aCSF) was slowly infused to cortical layers 3 and 4. After 3 weeks, the mice were used for the acute slice experiments.

Brain Slice Preparation
Mice were decapitated under deep anesthesia by inhaling 3% isoflurane in O 2 . The mouse brain was harvested and sliced using a vibratome (thickness ¼ 300 μm; VT1200S, Leica). During the slicing procedure, the immersion solution was the surgical aCSF solution kept at 4°C. Subsequently, the brain slices were incubated in the surgical aCSF solution at 35°C for 20 min and were immersed in the recording aCSF solution at room temperature (22°C to 24°C) with continuous aeration of 95% O 2 and 5% CO 2 for 30 min. The brain slices were mounted on an imaging chamber using a tissue anchor (SHD-41/10, Warner instruments). For the neuronal inhibition study, tetrodotoxin (TTX) was added to the recording aCSF at 10 μM. Damaged neurons were excluded based on their morphology if blebbing or a ruptured membrane was observed in a DIC image. Functional viability was subsequently confirmed using calcium responsiveness under optogenetic stimuli.

Optic Setup
Our customized optic system shown in Fig. 1(b) was designed to incorporate the following three modalities: (i) two-photon fluorescence imaging for recording neuronal activity; (ii) spectral reflectance spectroscopy for nanostructural readout of myelinated axons; and (iii) optogenetics for manipulating neural activity. The system was constructed based on an upright galvanometerbased laser scanning microscope (Ultima IntraVital, Bruker), coupled to a Ti-Sapphire femtosecond laser (for two-photon fluorescence imaging; Chameleon Ultra II, Coherent) and a supercontinuum white-light laser (for SpeRe and SCoRe; EXB-6, NKT photonics). The femtosecond laser was tuned to 920 nm for exciting GCaMP6s and was attenuated to 10 to 20 mW at the objective back aperture. The supercontinuum laser was attenuated to ∼0.4 mW at the objective back aperture using a neutral density filter and bandpass filtered to 450 to 700 nm. An apochromatic water-immersion objective lens (25×, 0.95 NA, Leica) was used for both two-photon fluorescence and SpeRe/SCoRe readouts. For two-photon fluorescence imaging, a GaAsP photomultiplier tube placed at the nondescanned path was used along with a bandpass filter at 500 to 550 nm. For the SCoRe imaging, a silicon photomultiplier tube placed at the descanned path was used. For SpeRe measurements, an array spectrometer (SR303i and Newton, Andor) was introduced at the descanned path. For spectroscopy, the grating of 600 lines · mm −1 was adjusted to accept a spectral window of 550 to 650 nm, where the input white light exhibited near uniform intensity profile over the spectral window. The slit size was set to 10 μm, providing a spectral resolution ∼0.13 nm with enough signal-to-noise ratio at the acquisition speed of 20 Hz. For optogenetics, a 633-nm diode laser (MRL-III-633, CNI laser) was coupled to a multimode fiber (400 μm core, 0.39 NA; M119L02, Thorlabs), which was mounted on a motorized three-axis micromanipulator (MP-285, Sutter Instrument). Optical irradiance for optogenetic stimulation was set to 10 mW · mm −2 at the tissue surface, which was delivered at 100 Hz with 50% duty cycle for the duration of 0.5 to 2 s.

Data Analysis
For SpeRe, the time-series reflectance spectra were filtered in the spectral and time domains in MATLAB. To reduce artifactual jittering noise, the low-pass filter at a cutoff frequency of 0.65 nm −1 was applied in the wavelength domain by applying the "low-pass" function, and the smoothing filter with the bin of 0.5 s was applied in the temporal domain using the "smooth data" function. From the filtered spectral data, the relative phase shift over time from the baseline was retrieved based on the least-squares method. Occasionally, unpredictable motion artifacts (e.g., instability of media perfusion) interfered reliable quantification of the phase shift. Thus we excluded the data if it displayed at least one of the following indications of excessive motion artifact: change in reflectance intensity >10% or the phase drifted over 3 nm. For calcium imaging data, we quantified relative change in fluorescence normalized by the baseline fluorescent intensity (ΔF∕F).

Statistical Analysis
GraphPad Prism was used for statistical analysis. Group comparisons were conduction using unpaired t-tests (parametric). The data are presented as mean ± standard error. We considered a p-value of <0.05 to be statistically significant.

System for All-Optical Investigation
To observe the morphological dynamics of functionally active myelinated axons at the nanoscale, we introduced a all-optical neurophysiology approach [Figs. 1(a) and 1(b)]. For manipulating neural excitation with minimal mechanical perturbation, we introduced red-shifted excitatory optogenetic protein (ChrimsonR) into cortical excitatory neurons. Neuronal excitation was timely triggered by an epi-illuminated fiber-coupled light-emitting diode (LED) at 633 nm on an acute brain slice and was confirmed by GCaMP-mediated functional calcium imaging on the targeted neurons by two-photon microscopy. 20 For recording nanostructural dynamics of myelinated axons, we incorporated spectral reflectometry (SpeRe) that captures broadband reflectance spectra at the geometric center of the axons and decodes the physical size of the multilayered thin films (i.e., diameter of the myelinated axons) by decoding the spectrums. To assist with pinpointing the geometric centers, we additionally introduced spectral confocal reflectance imaging (SCoRe), 21 which provides a volumetric image of reflected light corresponding to the centerlines of myelinated axons.
The overall procedure for nanostructural readout begins with acquisition of a volumetric reflectance image from a fresh brain slice by SCoRe [ Fig. 1(c)]. From the volumetric SCoRe image, the position of maximum intensity for each cross section is localized, resulting in the geometric centerline along the fibrous structure. The broadband reflectance spectrum is subsequently acquired over time at the center positions, and the structural dynamics is decoded from the acquired spectra (SpeRe).
For decoding of nanostructural dynamics, we performed numerical optic simulation on the myelinated axons and obtained quantitative relationship between the reflectance spectrum and the nanoscale cytoarchitecture. Briefly, the interaction of light waves at the subcellular layers of myelinated axons was described by the thin-film matrix theory, and the distribution of light waves at the focus was formulated by the vector diffraction theory. As the morphological dynamics were observed exclusively at the paranode of myelinated axons in our following experiments, we set the simulation parameters including physical size and refractive index for each subcellular layer based on the paranodal region (Table S1 in the Supplementary Material).
The resulting simulation database showed that the swelling of myelinated axons (ΔD > 0) leads to negative phase-shift in the wavenumber domain (Δv < 0) with linear relationship (Fig. S1 in the Supplementary Material). Thus we decided to use the relative phase shift (Δν) as a reliable metric to quantify the dynamic swelling of myelinated axons (i.e., phase detection method [22][23][24]. To detect subnanometer-scale morphological dynamics, we used a grating of 600 lines · mm −1 and a slit size of 10 μm, resulting in a spectral resolution of ∼0.13 nm.

Validation of Subnanometer-Scale Readout Precision
SpeRe was previously applied for measuring nanoscale morphological dynamics, such as osmotic swelling of myelinated axons or protein adhesion to a functionalized microsphere. 19,25 To further verify the subnanometer-scale precision of our SpeRe readout, we used a finely tapered glass fiber prepared by thermally drawing a glass rod using a micropipette puller (Fig. 2). In a tapered fiber, the diameter (D) is a smooth function of its longitudinal position (x); therefore, we can simply mimic the nanoscale change in diameter (ΔD) by shifting the position (Δx) using a galvanometric scanner. We first estimated the diameter along the fiber using an image acquired by polarization microscopy and validated by SpeRe [Figs. 2(a) to 2(c)]. We then selected two points, x 1 and x 2 , which showed physiological axon diameters (0.3 to 5 μm). By the linear regression near the selected points, we acquired the slope (dD∕dx), which provided the multiplicative factor for converting the step shift in position (Δx) to the change in diameter (ΔD). For example, to introduce an increase in diameter by 0.5 nm at x 2 , we shifted the position along the centerline by þ55 nm, corresponding to ΔD (0.5 nm) divided by dD∕dx (0.009).
In a tapered fiber, we obtained broadband reflectance spectra over time on the two selected positions at a sampling speed of 20 Hz. To convert the spectral shift (Δv) to the change in

Activity-Dependent Nanostructural Dynamics of Myelinated Axons
For optical manipulation and recording of neural excitation, we microinjected two types of adenoassociated viruses encoding a red-shifted opsin (AAV9-hSyn-ChrimsonR-tdTomato) and a fluorescent calcium indicator (AAV9-hSyn-GCaMP6s) at the somatosensory cortex of live mice and prepared fresh brain slices in oxygenized medium on the experiment day 26,27 [ Fig. 3(a)].
By combining two-photon fluorescence and confocal reflectance imaging of the brain slice, we visualized transfected neurons, as well as their myelinated axons. By illuminating 633-nm light pulses following the high frequency stimulation protocol, we observed reliable functional calcium activity in the most neuronal soma expressing both ChrimsonR and GCaMP6s 10,13 [ Fig. 3(b)   To address whether neuronal excitation leads to nanostructural dynamics of myelinated axons, we randomly sampled up to 10 myelinated axons for each brain slice and recorded the nanoscale dynamics by SpeRe for 5 s with light stimuli ["ChR + light" group, n ¼ 104 axons in 5 mice; Figs. 3(c) and 3(d)]. We specifically targeted the en passant axons in the periphery of the viral injection site due to their inherent sparsity. As negative control groups, we included brain slices without light stimuli ("ChR only" group, n ¼ 105 axons in 5 mice) and brain slices without introducing the optogenetic actuator ("light only" group, n ¼ 105 axons in 5 mice). The SpeRe readout was performed near either end of the myelin sheaths, corresponding to the paranode, where axomyelinic communication is known to be active. [28][29][30] Apparently, only the group with functional optogenetic excitation (ChR + light group) showed statistically significant spectral shift by ∼2000 cm −1 on average [unpaired t-test: p < 0.05; Fig. 3(e)]. Intriguingly, the spectral shift was cumulatively increased during the 1-s period of optogenetic excitation and slowly recovered in several seconds, indicating that the activity-dependent morphological dynamics does not follow the neuronal membrane potential having millisecond-scale rise and fall kinetics. Moreover, pharmacological inhibition of action potential generation by TTX significantly, but only partially, attenuated the swelling, suggesting that the generation of action potentials is not necessary for inducing the swelling (Fig. S4 in the Supplementary Material).   To convert the spectral shift (Δv) to the change in physical diameter (ΔD), we applied the linear relationship derived from our numerical simulation (Fig. S1 in the Supplementary Material). As the nanostructural parameters for individual myelinated axons that we sampled were not attainable, we applied the representative structural parameters in Table S1 in the Supplementary Material obtained from previous electron micrographs and estimated from our optical images (Fig. S3 in the Supplementary Material). 19,31 Although this approach compromised the precision of estimation on individual axons, we were able to gain information on the population distribution. Although statistically significant, the group-averaged spectral shift of ∼2000 cm −1 corresponds to ∼0.2 nm, which was at least several folds smaller than previously reported axonal swelling observed in cell culture and invertebrate systems [ Fig. 3(e)]. This discrepancy can be explained by our experimental design, which randomly samples myelinated axons in brain slices. Conceivably, a significant portion of long-projecting myelinated axons is expected to be severed during the tissue slicing procedure and contains insufficient optogenetic proteins due to the stochastic nature of viral transfection. Consequently, the large portion of the samples even in the ChR + light group might have been nonresponsive to optogenetic stimuli. Indeed, the histograms of change in diameters for the experimental and the negative control groups were largely similar with statistically significant differences observed only at the swelling > ∼ 0.5 nm [ Fig. 3 We thus questioned if we could observe reliable nanostructural dynamics in the myelinated axons that are morphologically intact and functionally active (Fig. 4). By two-photon imaging of neuronal morphology and functional calcium activity with optogenetic stimuli, we identified the three morphologically intact and functionally active myelinated axons, which were connected to the intact neuronal soma

Discussion
By combining the nanoscale readout based on spectral interferometry (SpeRe) and optogenetic manipulation of neural excitation, we report the first experimental evidence that myelinated axons exhibit activity-dependent subnanometer-scale morphological dynamics of ∼0.5 nm. We further reveal that the nanostructural change displays slow kinetics at the second scale and localized to the paranode. As conduction efficacy of neural information has steep dependence on the subcellular structure of the paranode, we expect that its nanostructural dynamics can serve as a regulatory mode of controlling conduction speed in the mammalian brains.
The functional consequence of the morphological remodeling at the paranode can be inferred by the periaxonal nanocircuit model of the myelinated axons. 10 According to this model, the periaxonal space is electrically conductive and the paranodes are only partially sealed, resulting in leaky propagation of neural information. Thus the swelling of the paranode can attenuate the leakage and accelerate the conduction speed. By the theoretical double cable model, 1-nm swelling at the paranode leads to ∼1.3% acceleration in conduction speed. Accordingly, the observed swelling of ∼0.5 nm is expected to increase the conduction speed by ∼0.7%. Consistently, Yamazaki et al. 29,32 reported a short-term increase in conduction speed by directly depolarizing the myelinating oligodendrocyte. In addition, the paranodal remodeling may also involve analog modulation of action potential waveform, 33 which potentially affects synaptic coupling 34 and network synchrony. 35 Furthermore, fields and Ni reported that unmyelinated axons swell in response to action potential firing, leading to nonvesicular release of adenosine triphosphate from axons through volume-activated anion channels, 36,37 suggesting potential transmittermediated interaction between axons and nearby glia. The physiological relevance of the paranodal remodeling on neural circuit function requires further investigation.
Our results reveal that the paranodal swelling occurs cumulatively and reversibly at the second scale, suggesting that the neuronal membrane potential having a millisecond-scale rise and fall kinetics is not the direct source of the swelling [Figs. 3(d) and 4(d)]. In addition, pharmacologic inhibition of voltage-gated sodium channel by TTX only partially attenuated the paranodal swelling (Fig. S4 in the Supplementary Material), suggesting that the generation of action potential is not necessary for inducing the swelling. Considering that ChrimsonR is a light-gated channel permeable to both sodium and calcium ions, 27,38 we expect that the calcium influx may play a key role in the paranodal swelling. In agreement with our thought, we observed that calcium transient was evoked by ChrinsonR-mediated optogenetics even with TTXmediated inhibition of the voltage-gated sodium channel. The partial decline in calcium activity was correlated with the attenuation of paranodal swelling, also supporting the causal role of calcium activity in the swelling. Further pharmacological inhibition of ion channel subtypes or use of ion-selective opsins will clarify the underlying molecular mechanism.
Although our results consistently suggest that the neuronal excitation leads to enlargement of myelinated axons at the paranode, which subcellular components are remodeled remains to be answered. Chéreau  Opto. swelling in the mouse brain. 15 Trigo and Smith also showed micron-scale axonal swelling following prolonged electric stimulation of the peripheral nerves. 28 As mature myelin sheaths do not typically exhibit axonal activity-dependent functional responses, we estimate that the axon at the node of Ranvier swells in response to ionic redistribution across the membrane and that the surrounding myelin is passively enlarged. Detailed multiparametric analysis based on the thinfilm model with a priori structural information on each subcellular composition may provide a solution to this question. Alternatively, subnanometer-scale super-resolution imaging techniques (e.g., MINFLUX 39 and SUSHI 40 ) will help to reveal further details in activity-dependent structural dynamics.

Summary
As neural activity involves rapid ion flux across the cell membranes, researchers have long tried to detect the accompanying nanoscale morphological dynamics. However, measuring the activity-dependent nanostructural dynamics in the living mammalian brain has been an enigma due to the technical limitations. By combining excitatory optogenetics and in situ nanoscale metrology based on spectral interference, we demonstrate the first direct observation that the mammalian axons exhibit transient activity-dependent swelling at the subnanometer scale.

Disclosures
The authors have no relevant financial interests in this article and no potential conflicts of interest to disclose.